CN114672507B

CN114672507B - Bacterial outer membrane vesicle capable of presenting multiple heterologous peptides or proteins, construction method and application thereof

Info

Publication number: CN114672507B
Application number: CN202210280548.0A
Authority: CN
Inventors: 叶邦策; 吕召勇; 沃静
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2022-03-22
Filing date: 2022-03-22
Publication date: 2024-04-16
Anticipated expiration: 2042-03-22
Also published as: CN114672507A

Abstract

The invention discloses a bacterial outer membrane vesicle capable of presenting a plurality of heterologous peptides or proteins, and a construction method and application thereof. The method comprises the steps that the membrane surface and the inner cavity of the bacterial outer membrane vesicle are simultaneously loaded with a plurality of heterologous peptides or proteins, the heterologous peptides or proteins anchored on the surface of the bacterial outer membrane vesicle are displayed on the surface of the outer membrane vesicle through linker fusion bacterial membrane protein expression, the heterologous peptides or proteins loaded on the inner cavity of the bacterial outer membrane vesicle are loaded in the inner cavity of the outer membrane vesicle through fusion protein signal peptide or protein truncated body expression crossing the bacterial inner membrane, and the efficient production of the bacterial outer membrane vesicle capable of presenting the heterologous peptides or proteins is realized through related gene knockout of gram-negative bacteria.

Description

Bacterial outer membrane vesicle capable of presenting multiple heterologous peptides or proteins, construction method and application thereof

Technical Field

The invention belongs to the technical fields of synthetic biology and immunology, and particularly relates to a bacterial outer membrane vesicle capable of presenting a plurality of heterologous peptides or proteins, and a construction method and application thereof.

Background

Outer membrane vesicles (Outer Membrane Vesicle, OMV) are natural non-replicating vesicles with a size of 40-200nm produced by the outer membrane of gram-negative bacterial cells, which are rich in outer membrane proteins, phospholipids and lipopolysaccharides, and the inner cavity contains nucleic acid substances, enzymes, virulence factors and other molecules derived from the parent strain, and play an important role in the information exchange between bacteria and bacteria or between bacteria and hosts.

The two natural advantages of outer membrane vesicles make them excellent in immune activation, and thus are applied to the fields of vaccine development and drug delivery. Firstly, the structural advantage of the outer membrane vesicles of 20-400nm enables the outer membrane vesicles to enter lymph nodes preferentially and then be taken up by antigen presenting cells for treatment, so that efficient immune activation is caused. Second, the outer membrane vesicle surface is enriched in pathogen-associated molecular pattern molecules, preserving the ability of the bacteria to activate innate and adaptive immunity. Outer membrane vesicles based on these two advantages have been developed for application in the biomedical field, especially in the field of vaccine development. Firstly, the outer membrane vesicle from pathogenic bacteria has specific antigen of parent bacteria, so the outer membrane vesicle can induce human body to generate specific immune response against pathogenic bacteria, thereby achieving the purpose of preventing diseases, for example, OMV vaccine from group B meningococcus is successfully marketed and applied in multiple countries, and excellent prevention effect is achieved. On the other hand, antigen or therapeutic protein, polypeptide or protein or polypeptide with targeting function can be loaded to OMV by genetic engineering means, so that OMV becomes antigen or drug delivery carrier for disease prevention and treatment, and the research and development stage is still in progress at present.

The current form of loading heterologous peptides or proteins on OMVs is largely divided into two types, surface display and luminal loading: the surface display is to fuse and express the heterologous peptide or protein with the anchor protein by taking the outer membrane protein as the anchor protein, the anchor protein targets and fixes the heterologous peptide or protein on the outer membrane surface so as to obtain the outer membrane vesicle with the heterologous peptide or protein displayed on the surface along with the generation of the outer membrane vesicle, but the limitation of the surface display on the type of the heterologous peptide or protein leads to the incapability of realizing the membrane surface display of the outer membrane vesicle by the heterologous peptide or protein with large partial mass and complex space structure. The method of lumen loading is to target the heterologous peptide or protein in the lumen of the outer membrane vesicle, and is generally achieved by fusion expression of the heterologous peptide or protein with a signal peptide or protein containing a signal peptide, wherein the fusion protein is first targeted to the periplasmic space, and as the outer membrane vesicle is produced, the protein in the periplasmic space is naturally encapsulated into the outer membrane vesicle, thereby achieving loading of the heterologous peptide or protein in the lumen. Since the prokaryotic periplasmic space provides the enzyme and oxidation conditions required for protein folding and disulfide bond formation, this greatly widens the variety of the outer membrane vesicle in which the inner cavity can be loaded with a heterologous peptide or protein, while the inner cavity position of the outer membrane vesicle can protect the heterologous peptide or protein from degradation by external factors, but the method of loading the heterologous peptide or protein in the inner cavity cannot realize a partial function of displaying the heterologous peptide or protein on the membrane surface, such as a targeting function.

Although the membrane surface or the inner cavity can realize the display or the loading of the heterologous peptide or the protein on the outer membrane vesicle, the prior art can not realize the simultaneous loading of a plurality of heterologous peptides or proteins on the outer membrane vesicle, so that the requirements of the outer membrane vesicle as a multivalent antigen delivery carrier and a multifunctional disease targeting treatment carrier can not be met.

Disclosure of Invention

In order to solve the difficult problem in the prior art, the invention breaks through the limitation of the bacterial outer membrane vesicle in the aspect of loading a plurality of heterologous peptides or proteins simultaneously, and aims to provide the bacterial outer membrane vesicle capable of presenting a plurality of heterologous peptides or proteins, and the construction method and application thereof.

In a first aspect, the present invention provides a bacterial outer membrane vesicle which can present a plurality of heterologous peptides or proteins, in particular an outer membrane vesicle having two heterologous peptide or protein loading modules, comprising an outer membrane vesicle membrane surface heterologous peptide or protein display module and an outer membrane vesicle lumen heterologous peptide or protein loading module.

A method for constructing a bacterial outer membrane vesicle capable of presenting a plurality of heterologous peptides or proteins, comprising the following steps: the heterologous peptide or protein anchored on the surface of the bacterial outer membrane vesicle is displayed on the surface of the outer membrane vesicle through linker fusion bacterial membrane protein expression, the heterologous peptide or protein loaded on the vesicle inner cavity of the bacterial outer membrane vesicle is expressed through fusion of protein signal peptide or protein truncated body crossing the bacterial inner membrane so as to be loaded on the inner cavity of the outer membrane vesicle, the protein signal peptide exists in a free state, and the protein truncated body exists in a limited anchoring state.

Further, the bacterial membrane protein is ClyA, ompA, ompC or OmpF, preferably ClyA protein.

Further, the protein signal peptide is OmpA signal peptide or LPP signal peptide, and the protein carrier short body is OmpA truncate or OmpC truncate, preferably OmpA truncate.

Further, the heterologous peptide or protein is a therapeutic peptide, protein or antigen with targeting function.

In a second aspect, the present invention provides a preferred method of constructing a recombinant gram-negative bacterium producing outer membrane vesicles simultaneously loaded with a heterologous peptide or protein, the recombinant gram-negative bacterium having a system capable of expressing a plurality of heterologous peptides or proteins;

as a preferred embodiment, the present invention provides a method for constructing an engineering bacterium for efficient production of foreign peptide or protein-loaded outer membrane vesicles:

constructing a heterologous peptide or protein arabinose-induced expression system by using a pBAD plasmid to control the expression of a fusion protein of an outer membrane vesicle surface display module, wherein the fusion protein consists of a membrane protein and the heterologous peptide or protein; the constitutive expression system is located in the genome and the heterologous peptide or protein is located in the inner cavity of the outer membrane vesicle by means of fusion expression of the secretory protein signal peptide or secretory protein truncate and the heterologous peptide or protein.

In a third aspect, the present invention provides a preferred method of producing outer membrane vesicles simultaneously loaded with a plurality of heterologous peptides or proteins;

(1) Fusion expression of the heterologous peptide or protein and the ClyA gene by using a constructed plasmid is realized through a linker to be used as an expression system for outer membrane vesicle membrane surface display;

(2) Knocking out gram negative bacteria nIpl genes by utilizing Red/ET homologous recombination technology, and leading to high yield of outer membrane vesicles, thus obtaining a strain with high yield of outer membrane vesicles;

(3) Inserting a heterologous peptide or protein gene sequence into the downstream of the genome coding OmpA membrane protein gene sequence of the strain obtained in the step (2) by utilizing a Red/ET homologous recombination technology on the basis of the step (2), and replacing the periplasmic part of the OmpA protein with the heterologous peptide or protein to obtain an engineering strain;

(4) Transferring the plasmid which is constructed in the step (1) and is responsible for displaying the heterologous peptide or protein system on the surface of the outer membrane vesicle membrane into the engineering strain constructed in the step (3) to obtain recombinant gram-negative bacteria which are produced efficiently and simultaneously load a plurality of heterologous peptide or protein outer membrane vesicles;

(5) Inoculating the engineering bacteria constructed in the step (4) into a liquid culture medium for culturing for a period of time, then adding a plasmid expression inducer, wherein the inducer is an arabinose inducer, and then continuing overnight culture;

(6) Centrifuging the cultured bacterial liquid, collecting supernatant and filtering by using a sterile filter head; concentrating the filtered supernatant with ultrafiltration tube, and extracting outer membrane vesicle in concentrated solution with outer membrane vesicle extraction kit or density gradient centrifugation.

Further, the recombinant gram-negative bacterium can secrete outer membrane vesicles with high efficiency, and the characteristics are realized by knocking out related genes, wherein the related genes are tolA genes or nIpl genes, and preferably the nIpl genes.

Preferably, the culture conditions in step (5) are 37℃at 220rpm,

preferably, the centrifugation conditions in step (6) are 4℃at 12000rpm,

preferably, the final arabinose-induction concentration of step (5) is 30mm,

preferably, the sterile filter head of step (6) has a pore size of 0.22 μm,

preferably, the ultrafiltration tube of step (5) has a molecular weight cut-off of 100kDa.

The application of the bacterial outer membrane vesicle capable of presenting a plurality of heterologous peptides or proteins in the preparation of vaccines can lead the bacterial outer membrane vesicle to simultaneously load a plurality of heterologous peptides or proteins on the membrane surface and in the cavity when being used as an infectious disease prevention vaccine or a tumor vaccine, widens the loading quantity and types of the heterologous peptides or proteins, and can be rapidly absorbed by immune cells in lymph nodes under the condition of loading the heterologous peptides or proteins due to the nano-structure advantage and the nature adjuvant property, thereby leading to targeted immune response and achieving the effects of preventing infectious diseases or treating tumor immunity.

Compared with the prior art, the invention has the beneficial effects that:

1) The outer membrane vesicle loaded with a plurality of heterologous peptides or proteins can simultaneously allow the heterologous peptides or proteins to be loaded on the outer membrane vesicle, and the inner cavity allows the heterologous peptides or proteins with complex space structures and larger mass to be loaded, so that the limitation of the size of the displayed heterologous peptides or proteins on the surface of the outer membrane vesicle and the possibility of the outer membrane vesicle used as a multivalent vaccine carrier are expanded, and the technical foundation of long-distance delivery of a therapeutic substance after drug delivery or tumor targeted therapy and gastrointestinal tract absorption of the outer membrane vesicle is also expanded; the recombinant gram-negative bacteria can efficiently produce outer membrane vesicles loaded with a plurality of heterologous peptides or proteins, and a thought is provided for deep transformation of the outer membrane vesicles;

2) In the invention, the combined delivery (NR-OMV) of novel coronavirus antigens NG06 and RBD antigens is taken as an example, and a mouse experiment proves that after single needle administration for 10 days, the detection of the serum antibody titer of the mouse shows the antibody response level superior to that of the traditional protein matching adjuvant immunization method, and after intraperitoneal administration for 30 days, the antibody titer is higher, and compared with the single antigen administration groups (OMV-NG 06 group and OMV-RBD group), the NR-OMV shows the antibody titer at a higher level, so that the NR-OMV immunization effect is better than that of the single antigen immunization and the traditional protein matching adjuvant immunization method.

Drawings

FIG. 1 is a schematic representation of NR-OMVs;

FIG. 2 is a diagram showing verification of nIpl gene knockout;

FIG. 3 is a diagram showing the genome insertion verification of RBD antigen gene sequences;

FIG. 4 is a Western blotting graph of outer membrane vesicle loaded NG06 and RBD antigen;

FIG. 5 is a NR-OMV transmission electron microscope topography;

FIG. 6 is a graph comparing uptake of outer membrane vesicles by dendritic cells;

FIG. 7 is cytokine quantification after stimulation of immune cells by wild-type outer membrane vesicles and outer membrane vesicles loaded with heterologous peptides or proteins;

FIG. 8 shows high expression of surface co-stimulatory factors after stimulation of immune cells by wild-type outer membrane vesicles and outer membrane vesicles loaded with heterologous peptides or proteins;

FIG. 9 shows the detection of RBD antibody production levels by mouse serum ELISA.

Detailed Description

For a better understanding of the objects, technical solutions and advantages of the present invention, the present invention will be described in detail with reference to the following examples and accompanying drawings. The specific embodiments described herein are merely illustrative of the invention and are not intended to limit the practice of the invention.

Embodiment case one: preferred method for constructing engineering strain capable of highly producing outer membrane vesicles loaded with a plurality of heterologous peptides or proteins

1. EcN-pKD46 electrotransformation competent cells were prepared with E.coli Nissle 1917 as original strain:

1.1 transferring pKD46 plasmid into EcN competent cells to obtain EcN-pKD46 (culture temperature: 30 ℃);

1.2 inoculating EcN-pKD46 to 50ml LB culture medium, culturing at 30deg.C and 220rpm until OD=0.15, adding arabinose inducer with a final concentration of 30mm to induce expression of 3 enzymes of pKD46 plasmid, and continuing culturing until OD=0.4;

1.3 taking out the bacterial culture bottle, carrying out ice bath for 30min, and centrifugally collecting thalli at 4 ℃ and 3500 rpm;

1.4 re-suspending with precooled sterile water, centrifuging, removing supernatant, repeatedly cleaning once, and centrifuging again;

1.5 re-suspension with pre-chilled 10% sterile glycerol and centrifugation;

1.6 removing supernatant, adding 1ml of pre-cooled 10% sterile glycerol, packaging into 1.5ml EP tubes, 100 μl each, and storing in a refrigerator at-80deg.C.

2. Preparation of gene fragments of targeted knockout nIpl genes:

2.1, firstly amplifying by using a PCR technology and using a EcN genome as a template to obtain 500bp upstream of an nIpl gene and 500bp downstream of the nIpl gene, and amplifying by using a pSET152 plasmid as a template to obtain an apramycin resistance gene, and performing gel cutting on a nucleic acid electrophoresis gel to recover a gene fragment;

2.2, connecting the three recovered gene fragments by using an overlay PCR technology, performing gel cutting recovery on a PCR product twice after nucleic acid electrophoresis to ensure the purification effect, and finally obtaining a PCR targeting fragment for knocking out the nIpl gene for transforming and knocking out the nIpl gene;

3. gene fragment transformation and verification of knockout nIpl gene

3.1PCR targeting gene fragment electrotransformation (1900V, 5-6 ms) into EcN-pKD46 electrotransformation competent cells, coating on solid LB culture medium plates with double resistance of apramycin (50 ng/. Mu.l) and ampicillin (100 ng/. Mu.l), and placing in a bacterial incubator at 30 ℃ for culturing and growing for 16 hours;

3.2, selecting monoclonal and screening positive clones by colony PCR technology;

4. preparing EcN-pKD46.DELTA.nIpl into electrotransformation competent cells for use;

5. preparing, transforming and verifying RBD targeting gene fragments inserted into genome in a targeted manner:

5.1, amplifying by taking a synthesized S protein gene sequence as a template to obtain RBD gene fragments, amplifying by taking a pKD3 plasmid as a template to obtain cm resistance genes, and amplifying by taking a EcN genome as a template to obtain OmpA upstream and downstream 500bp gene fragments;

5.2 obtaining a targeting gene fragment containing the RBD target sequence for genome insertion by using an overlay PCR technology;

5.3, the targeting gene fragment is transferred into EcN-pKD46 delta nIpl competent cells by an electrotransformation method, coated on a solid LB culture medium plate with chloramphenicol (25 ng/. Mu.l) and ampicillin (100 ng/. Mu.l) double resistance, and placed in a bacterial incubator at 30 ℃ for culturing and growing for 16 hours;

5.4 picking single colony, verifying by colony PCR technology, and using the positive strain EcN-pKD46-R delta nIpl for downstream experiments;

5.5 Positive strain EcN-pKD 46-NR.DELTA.nIpl was cultured at 37℃and 220rpm for 4-5 passages to eliminate pKD46 temperature-sensitive plasmid to obtain strain EcN-R.DELTA.nIpl, and EcN-R.DELTA.nIpl electrotransformation competent cells were prepared.

6. Construction of expression plasmid:

6.1 amplifying by using a PCR technology and taking a synthesized S protein gene sequence as a template to obtain a NG06 fragment, amplifying by using a MG1655 genome as a template to obtain a ClyA gene fragment, and cutting gel for recovery after nucleic acid electrophoresis.

6.2 digesting the pBAD-24 plasmid by using NcoI and HindIII endonuclease, and cutting gel and recovering enzyme cutting products after nucleic acid electrophoresis;

the enzyme digestion preferred method comprises the following steps:

6.3 constructing plasmids by using a multi-fragment one-step rapid cloning kit, and carrying out water bath for 30min at 50 ℃, wherein the system comprises the following steps:

composition of the components	Volume of
		pBAD-24 linear plasmid	4μl
ClyA gene fragment	1μl
		GFP gene fragment	1μl
2 x same asSource recombinant enzyme	10μl
		H2O	4μl

6.4 transformation into DH 5. Alpha. Chemically competent cells;

6.4.1 taking DH5 alpha chemically competent cells out of the refrigerator at-80 ℃ and thawing on ice;

6.4.2 adding 10. Mu.l of the homologous recombination reaction solution to DH 5. Alpha. Chemically competent cells, slightly shaking and then carrying out ice bath for 30min;

6.4.3 heat shock in a water bath at 42 ℃ for 90s, and placing on ice for 2min;

6.4.4 adding 450 μl LB medium, culturing at 37deg.C and 200rpm for 45min;

6.4.5 evenly spread on an ampicillin antibiotics plate, and cultured overnight in an incubator at 37 ℃;

6.5, screening positive clones by colony PCR the next day, shaking bacteria, and extracting plasmids;

pBAD-ClyA-NG06 transfer to EcN-RΔnIpl

Electrotransformation of the constructed pBAD-ClyA-NG06 plasmid into EcN-RΔnIpl electrotransformation competent cells, pre-culture, coating on an ampicillin flat plate, culturing overnight at 37 ℃, screening positive single colonies by colony PCR every other day to obtain EcN-NRΔnIpl strain and preserving the strain at-80 ℃ by glycerol;

the pBAD plasmid arabinose expression system is used for controlling the expression of heterologous peptide or protein anchoring on the surface of the outer membrane vesicle, RBD genes are inserted into a proper position at the downstream of the OmpA of a genome by a Red/ET technology (figure 3), RBD antigens are anchored in the inner cavity of the outer membrane vesicle by utilizing the N-terminal part of the OmpA protein, so that the anchoring (NR-OMV) of NG06 and the RBD antigens is completed, and likewise, the Red/ET technology is used for knocking out the nIpl genes (figure 2), the genes can cause the decrease of the contact effect between the outer membrane of bacteria and peptidoglycan and the inner membrane, and indirectly cause the increase of the fluidity of the outer membrane of bacteria, so that the engineering bacteria can produce the outer membrane vesicle with high yield.

Implementation case two: extraction method of NR-OMV

1. Engineering bacteria EcN-NR.DELTA.nIpl were streaked on LB medium plates with double resistance to chloramphenicol (25 ng/. Mu.l) and ampicillin (100 ng/. Mu.l), and cultured in an incubator at 37℃for 12 hours;

2. the next day, picking up the monoclonal and inoculating to 5ml LB culture, and culturing for 12 hours at 220rpm based on 37 ℃;

3. inoculated at 1% to 200ml of LB medium to OD=0.5, added with arabinose at a final concentration of 30mm, followed by culture at 37℃for 18h at 220 rpm.

4. Collecting bacterial culture supernatant at 12000rpm with a pre-cooled centrifuge at 4deg.C, filtering the supernatant with a 0.45 μm filter head, concentrating with a 100kDa ultrafiltration tube, concentrating to 10ml, extracting with an outer membrane vesicle extraction kit, measuring protein concentration with BCA method, and storing at-80deg.C.

5. Taking a proper amount of modified OMV sample for western blotting experiment verification, taking wild OMV as a control, and detecting anchoring of NG06 and RBD antigens in outer membrane vesicles by using SARS-COV-2-RBD rabbit polyclonal antibody;

TEM observations of outer membrane vesicle morphology: taking 10 mu l of outer membrane vesicles which are filtered by a 0.45 mu m sterile filter head and have the concentration of 0.6mg/ml, dripping the outer membrane vesicles into a copper mesh used for TEM observation, dripping 20 mu l of negative dye liquor (5% ammonium molybdate) when a sample is in a semi-dry state after 5min, and observing the sample on a machine after airing;

the loading of NG06 and RBD on NR-OMV is verified by western blotting experiments (figure 4), TEM observes that the appearance of the outer membrane vesicle is a bubble structure (figure 5), the construction of the outer membrane vesicle presenting a plurality of heterologous peptides or proteins successfully expands the way that the outer membrane vesicle is used as a multivalent vaccine or a combined vaccine or a drug delivery platform, the oxidation environment of the periplasmic space of bacteria provides advantages for protein folding and is easy to display larger heterologous peptides or proteins, and the problems that the quantity of the heterologous peptides or proteins anchored by a genetic engineering method is small are solved at the same time when the inner membrane and the outer membrane of the outer membrane vesicle are displayed; the outer membrane vesicle is used as a high-quality nanoparticle with natural adjuvant property, has huge development potential in the foreseeable future, is indispensable to the deep engineering strategy and technology of OMVs, and provides a new method for constructing the outer membrane vesicle presented by a plurality of heterologous peptides or proteins in the aspect of using the OMVs as biological prevention and treatment carriers.

Third embodiment of the invention the use of outer membrane vesicles loaded with a plurality of heterologous peptides or proteins as novel coronavirus antigen delivery vehicles

1. Dendritic cells (DC 2.4) ingest outer membrane vesicles (NR-OMVs):

1.1WT-OMV, NR-OMV were incubated with DiD dye (1:500) for 30min, respectively, and unbound DiD dye was rinsed off with a 100kDa ultrafiltration tube;

1.2 at 3X 10 ⁴ Cell density per well DC2.4 cells were seeded in 24 well plates, after incubation adherence, WT-OMV (DiD) was added, NR-OMV (DiD) incubated for 4h, DC2.4 cells were collected, centrifuged, and the cells were washed three times with PBS and examined with flow cytometry;

2. expression of outer membrane vesicles (NR-OMVs) stimulated dendritic cells (BMDCs)

BALB/c mouse bone marrow primary cells were taken and induced to differentiate into immature BMDC cells in vitro using GM-CSF (20 ng/ml) and IL-4 (10 ng/ml); incubating PBS, RBD, WT-OMV, NR-OMV, LPS and immature BMDC cells for 8h respectively, collecting suspension cells and cell culture supernatant respectively, washing stimulated cells with PBS, incubating the stimulated cells with MHCII direct fluorescent antibody (FITC) and CD86 direct fluorescent Antibody (APC) for 60min, washing with PBS for three times, and analyzing with a flow cytometer; detecting the content of IL-6 and TNF-alpha in the cell culture supernatant by using an ELISA kit;

by comparing the uptake of WT-OMVs with NR-OMVs by DC2.4 cells, engineering of outer membrane vesicles did not affect the uptake of outer membrane vesicles by dendritic cells (fig. 6), which suggests that OMVs significantly stimulated BMDC cell surface MHC II and CD86 molecules were significantly highly expressed (fig. 8), while secretion of cytokines IL-6 and TNF- α also aided in verifying this conclusion (fig. 7), outer membrane vesicles presenting multiple heterologous peptides or proteins had the same immune system stimulating effect as wild-type OMVs, and engineering of genes did not affect the adjuvant properties of OMVs.

NR-OMV stimulated mice to develop specific antibody manifestations

Preparing 30 BALB/c mice of 6-8 weeks, and dividing the mice into 6 groups, wherein 5 mice in each group are respectively PBS group, RBD+Alum positive control group, NG06-OMV single antigen immune group, RBD-OMV single antigen immune group and NR-OMV immune group; on days 0, 10, 20 of the experiment, PBS (150. Mu.l), RBD+Alum (2. Mu.g), NG06-OMV (10. Mu.g), RBD-OMV (10. Mu.g), NR-OMV (10. Mu.g) were injected intraperitoneally, respectively; collecting venous blood of the mice before the second immunization and 10 days after the third immunization, sorting, and performing ELISA detection analysis on serum;

the results showed that the serum of the NG06-OMV experimental group, the RBD-OMV experimental group, the NR-OMV experimental group, and the RBD+Alum experimental group were 1: the presence of RBD antibodies was detected at 100 dilutions (FIG. 9A), and the antibody levels were expressed as NR-OMV > RBD-OMV > RBD+Alum > NG06-OMV, with the antibody titer levels boosted after 3 immunizations at 1: the NR-OMV still shows the strongest antibody titer level under the 1000 dilution condition (figure 9B), which shows that both the internal and external antigens of the NR-OMV have immune efficacy, the combined display effect is better than the single antigen display effect, and the NR-OMV immune effect is obviously better than the RBD+Alum traditional vaccine immune effect.

The above description has been made generally for an outer membrane vesicle presenting a plurality of heterologous peptides or proteins, and applications thereof in disease prevention and treatment, and engineering bacteria producing the outer membrane vesicle, and specific construction processes and embodiments are described in detail, and it should be noted that modifications and perfections made on the basis of the embodiments can also achieve the effects of the present invention, so modifications and perfections made on the basis of the claims of the present invention are all within the scope of the claims of the present invention.

Claims

1. A method for constructing a bacterial outer membrane vesicle capable of presenting a plurality of heterologous peptides or proteins, comprising the following steps: the heterologous peptide or protein anchored on the surface of the bacterial outer membrane vesicle is displayed on the surface of the outer membrane vesicle through linker fusion bacterial membrane protein expression, the heterologous peptide or protein loaded on the inner cavity of the bacterial outer membrane vesicle is expressed through fusion of protein signal peptide or protein truncated body crossing the bacterial inner membrane so as to be loaded on the inner cavity of the outer membrane vesicle, the protein signal peptide exists in a free state, and the protein truncated body exists in a limited anchoring state;

the bacterial membrane protein is ClyA, ompA, ompC or OmpF;

the protein signal peptide is OmpA signal peptide or LPP signal peptide, and the protein carrier short body is OmpA truncated body or OmpC truncated body.

2. The method of claim 1, wherein the heterologous peptide or protein is a therapeutic peptide, protein, or antigen with targeting function.

3. The method of claim 1, wherein the bacterial membrane protein is ClyA protein and the protein carrier is OmpA truncate.